In the past, bacteria separated from the natural environment or artificial mutants of such bacteria have been used as microorganisms for the production of L-threonine by fermentation. Many L-threonine-producing artificial mutants are known, the majority of which are resistant to .alpha.-amino-.beta.-hydroxyvaleric acid and belong to the genus Escherichia, Serratia, Brevibacterium or Corynebacterium. Regarding Escherichia, in JPA 55-131397, JPA 59-31691, JPA 56-15696 and JPW 3-501682 are there described methods for the production of L-threonine by bacteria transformed by recombinant plasmids containing threonine operons.
Also, of the heretofore known threonine-producing bacteria, Escherichia coli BKIIM (VKPM) B-3996 strain has exhibited the most superior level of threonine production and coefficient of consumption (JPW 3-501682). According to the present invention, "coefficient of consumption" refers to the number of grams of sugar required for the production of one gram of threonine. If this bacterium is cultured in an experimental fermenter, adding sugar-ammonia to the culture medium in response to the signals from the pH sensor, then the maximum degree of biosynthesis of threonine is 85 g/l, and the coefficient of consumption is 2 g of sugar per one gram of threonine.
Aspartokinase (hereunder abbreviated to AK) is an enzyme which converts aspartic acid to .beta.-phosphoaspartic acid, and it is the main regulatory site of the biosynthesis pathway of aspartic acid and its derivative amino acids. As shown in FIG. 1, there are three types of AK from E. coli (AK I, AK II, AK III) and the first two of these are bifunctional enzymes having homoserine dehydrogenase (hereunder sometimes abbreviated to HD) activity. One of these is AK I-HD I which is coded for by the thrA gene, and the other is AK II-HD II which is coded for the metL(M) gene.
Only AK III is a monofunctional enzyme, and it is the product of a gene named lysC, and is known to undergo repression and feedback inhibition by lysine. On the other hand, AK I undergoes concerted repression by threonine and isoleucine, and inhibition by threonine, while AK II undergoes repression by methionine. The proportion of the intracellular activities thereof is AK I:AK II:AK III=approximately 5:1:4.
The lysC gene of E. coli has already been cloned, and its base sequence has been determined (Cassan, M., Parsot, C., Cohen, G. N. and Patte. J. C., J. Biol. Chem., 261, 1052, 1986). Also, the production of L-threonine by fermentation using strains of E. coli whose AK III activity has been reinforced, is described by Mizukami, et al. (Mizukami, T. et al., Agric. Biol. Chem., 50, 1015, 1986). However, sufficient release of the lysine-dependent feedback inhibition on the AK III reported by Mizukami, et al. has not yet been achieved.
Thus, the subject matter of the present invention is obtaining AK III with sufficient release of the feedback inhibition due to lysine, and providing a method for the production of L-threonine by fermentation which is much improved over the prior art.